DESCRIPTION (Applicant's abstract): The long-term goal of this project is to unravel the complex redox regulation of calcium flux and homeostasis, and thus of excitation-contraction (E-C) coupling, in muscle. Our recent findings have concentrated our focus on the intracellular calcium release channel of the sarcoplasmic reticulum (SR) of skeletal muscle, the type 1 ryanodine receptor (RyR1), and its regulation by nitric oxide (NO). RyR1 is essential for E-C coupling. We have discovered that 6-8 of 101 cysteines in this channel protein comprise an "oxygen sensor": the redox state of these residues is modulated reversibly by O2 tension, almost certainly via a redox enzyme system in the SR. Additionally, we have found that at physiological O2 tension (about 10 mm Hg), nanomolar concentrations of NO activate this channel by S-nitrosylation of a single cysteine thiol, and that this effect is dependent on another endogenous modulator, calmodulin. In other words, O2 tunes the response to NO, which in turn is transduced by calmodulin. The specific aims of this proposal are to: 1a. Characterize the behavior of the RyR1 oxygen sensor and the activation of RyR1 by NO over the physiologically relevant range of tissue oxygen tension. 1b. Identify the reversible oxidative modification of cysteines that comprise the oxygen sensor and localize within RyR1 those residues and the critical cysteine that is dynamically S-nitrosylated. 2 Characterize in vitro the functional consequences of replacing identified RyR1 cysteines by site-directed-mutagenesis. 3. Characterize the enzyme system in the SR that dynamically controls the redox state of RyR1. 4a. Determine the effects of exogenous and endogenously generated NO on depolarization-induced cytosolic Ca2+ flux in intact skeletal muscle fibers over the physiologically relevant range of PO2. 4b. Re-examine the effects of endogenously produced NO and reactive oxygen species on whole muscle contractility under physiologically relevant O2 tensions. The completion of these aims will provide new insights into the regulation of Ca2+ homeostasis in muscle and may allow a better understanding of disease states such as diaphragmatic dysfunction, malignant hypothermia and heat stroke in which the ryanodine receptor plays a central role.